Xylarichalasin A, a Halogenated Hexacyclic Cytochalasan from the

6 days ago - A cytochalasan, xylarichalasin A, was obtained from the endophytic fungus Xylaria cf. curta harbored in Solanum tuberosum. Its structure ...
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Xylarichalasin A, a Halogenated Hexacyclic Cytochalasan from the Fungus Xylaria cf. curta Wen-Xuan Wang, Xinxiang Lei, Yan-Ling Yang, Zheng-Hui Li, Hong-Lian Ai, Jing Li,* Tao Feng,* and Ji-Kai Liu* School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, Hubei 430074, PR China

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S Supporting Information *

ABSTRACT: A cytochalasan, xylarichalasin A, was obtained from the endophytic fungus Xylaria cf. curta harbored in Solanum tuberosum. Its structure was elucidated by comprehensive spectroscopic methods including HRESIMS, 1D/2D NMR, and residual dipolar coupling analysis as well as quantum chemistry calculations including DFT GIAO 13C NMR and ECD calculation. It has an unprecedented 6/7/5/6/6/6 fused polycyclic structure. In bioassay, xylarichalasin A showed cytotoxicity against human cancer cell lines with IC50 value ranging from 6.3−17.3 μM.

C

ytochalasans make up a group of fungal products majorly produced by genera Phoma, Helminthosporium, Penicillium, Aspergillus, Zygosporium, Metarhizum, Chaetomium, Rosellinia, Ascochyta, Hypoxylon, and Xylaria.1 For biosynthesis, their core structures are cyclized from a polyketide backbone. Their fusion patterns and substitution groups are highly variable, leading to rich structural diversity.2 Cytochalasans have a broad range of bioactivities including cytotoxic, antimicrobial, antiparasitic, phytotoxic, and antiviral activities.1,2 Recently, their fascinating aspects of structure and bioactivity have attracted many efforts on new structure discovery, biosynthesis, and chemical synthesis.3 Xylaria species are a rich source of bioactive secondary metabolites including 10-phenyl cytochalasans.4 Endophytic fungus Xylaria cf. curta was isolated from the health tissue of Solanum tuberosum.3b,c In our previous research on this strain, novel bioactive cytochalasans (curtachalasins A−E) were characterized.3b,c Thus, we kept investigating the cytochalasans produced by this strain in multiple fermentation conditions with diversified parameters including temperature, fermentation duration, supplement of metal salts, and nutrient composition. From the fermented medium with an optimized condition, xylarichalasin A was isolated with extensive chromatographic methods. Despite many attempts of obtaining single crystal turned to failure, its structure was assigned by HRESIMS, 1D/2D NMR, GIAO 13C NMR calculation, ECD calculation, and residual dipolar couplings (RDC) analysis with full confidence. Xylarichalasin A (1) has a 6/7/5/6/6/6 fused ring system with two chlorine substitutions, which features a new class of cytochalasan (Figure 1). Herein, we present its structural © XXXX American Chemical Society

Figure 1. Structure of xylarichalasin A.

Figure 2. 1H−1H COSY, key HMBC, and NOE correlations of 1.

identification, cytotoxic assay, and plausible biosynthesis pathway. Xylariachalasin A (1) was obtained as colorless amorphous powder with molecular formula determined to be Received: July 22, 2019

A

DOI: 10.1021/acs.orglett.9b02552 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Table 1. 1H NMR (600 MHz, J in Hz) and 13C NMR Data (150 MHz) of 1 (in Pyridine-d5) 1

no. 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 OAc

H NMR

2.70 2.13 1.57 1.90 2.53

(overlapped) (overlapped) (m) (m) (m)

2.70 0.81 5.01 2.27 2.33 2.10

(overlapped) 2.21 (dd, 11.5, 11.5) (d, 6.5) (dd, 10.8, 10.8) (dddd, 10.8, 10.8, 10.8, 3.3) (ddd, 10.8, 3.3, 3.3) 1.72 (ddd, 10.8, 10.8, 10.8) (m)

4.02 3.13 5.96 2.43 0.95

(d, 10.8) (ddd, 10.8, 10.8, 2.0) (d, 2.0) (s) (d, 6.6)

7.29 7.22 7.31 7.79

(overlapped) (overlapped) (overlapped) (dd, 7.6, 1.2)

1.79 (s)

Table 2. GIAO 13C NMR Calculation of Structure 1a and 1b

13

C NMR 178.0 139.0 45.7 49.9 30.9 34.7 37.8 55.2 101.0 37.5 20.1 69.8 42.2 33.9 36.3 85.2 214.9 73.5 42.2 70.3 27.0 15.5 138.8 138.2 129.5 127.5 127.1 127.9 170.1 20.0

C30H35NO6Cl2 by HRESIMS at m/z 598.1732 ([M + Na]+, calcd for 598.1734). The presence of two chlorine atoms was confirmed by the intensity of the isotope peak C30H35NO637Cl35ClNa+. The 1H, 13C NMR, and DEPT spectroscopic data (in pyridine-d5, Table 1) of 1 showed four methyl groups (δC = 15.5, 20.0, 20.1, 27.0 ppm), three methylene groups (δC = 33.9, 34.7, 37.5 ppm), ten sp3 methine groups (δC = 30.9, 36.3, 37.8, 42.2, 42.2, 45.7, 49.9, 69.8, 70.3, 73.5 ppm), four sp2 methine groups (δC = 127.1, 127.5, 127.9, 129.5 ppm), two sp3 quaternary carbons (δC = 55.2, 85.2 ppm), and seven sp2 quaternary carbons (δC = 101.0, 138.2, 138.8, 139.0, 170.1, 178.0, 214.9 ppm). The 1H−1H COSY experiment showed two spin−spin systems, namely H-4/H5(H2-11)/H-6(H3-12)/H2-7/H-8/H-13/H-14(H2-15/H-16/ H3-23)/H-20(H-19)/H-21 and H-26/H-27/H-28/H-29 (Figure 2). The vicinal J3 coupling constants between H-26/H-27/ H-28/H-29 were all 7.5 Hz (in CD3OD, Table S1), which indicated the presence of a double substituted phenyl group. The 6/7/5/6/6/6 fused ring system of 1 was further established by HMBC correlations (Figure 2). The 13C NMR chemical shifts (in CD3OD, Table S1) of the ring F were close to the reported compound curtachalasin A,3b which indicated that they share the same structure on this moiety. Therefore, the only downfield shifted CH-13 group (δH = 5.01, δC = 69.8 ppm) was supposed to be connected with chlorine. Since there was a benzene structure and one nitrogen connected double bond carbon, the remaining quaternary carbon (C-10) was deduced to bear another

no.

exptl. δCa

calcd δCb 1a

deviation

calcd δCb 1b

deviation

1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 OAc

176.2 137.9 45.0 50.1 30.7 33.6 37.2 54.3 103.2 37.4 20.1 67.4 41.1 32.8 35.9 83.5 212.8 72.7 42.0 70.5 25.7 14.7 136.7 135.4 129.1 127.5 127.0 127.7 171.2 20.5

174.6 139.2 45.0 51.2 30.8 32.9 38.4 56.5 100.0 37.8 19.6 66.1 41.3 32.7 37.0 85.2 215.3 71.6 43.3 70.7 23.1 15.1 137.8 137.5 128.2 126.4 126.0 128.7 167.8 20.0 MAE RMS sDP4+

−1.6 1.3 0.0 1.1 0.1 −0.7 1.2 2.2 −3.2 0.4 −0.5 −1.3 −0.1 −0.1 1.1 1.7 2.5 −1.1 1.3 0.2 −2.6 0.4 1.1 2.1 −0.9 −1.1 −1.0 1.0 −3.4 −0.5 1.19 1.48 99.82%

174.8 139.9 44.3 48.9 29.6 32.7 39.8 57.2 99.9 35.8 20.3 65.8 41.7 33.5 37.4 85.3 215.1 71.9 43.5 70.8 23.5 15.6 137.7 137.8 128.0 126.3 126.0 128.6 167.8 20.4 MAE RMS sDP4+

−1.4 2.0 −0.7 −1.2 −1.1 −0.9 2.6 2.9 −3.3 −1.6 0.2 −1.6 −0.6 0.7 1.5 1.8 2.3 −0.8 1.5 0.3 −2.2 0.9 1.0 2.4 −1.1 −1.2 −1.0 0.9 −3.4 −0.1 1.44 1.67 0.18%

Data recorded in CDCl3. bCalcd. δC were further scaling corrected after the conversion from calculated shielding tensors via formula δC (ppm) = 201.8−1.044 Icorr. a

Figure 3. Two possible relative configurations of 1.

chlorine. The spin−spin coupling constants of H-13, H-14, and H-20 suggested that they are at axial position. The coupling constant between H-8/H-13 was 10.8 Hz, suggesting H-8 is also at axial position. The spin−spin coupling splitting of Hax15 (J = 10.8, 10.8, 10.8 Hz) was induced by H-14, Heq-15, and H-16, and the coupling constant between Hax-15 and H-16 was deduced to be 10.8 Hz, which indicated that H-16 is at axial position as well. The coupling constant between H-21 and H20 was 2.0 Hz, proving that H-21 is an equatorial proton. NOEs were observed between H-5/H-8, H-4/H-21, H-16/H19, and H-19/H3-22, which determined the fusion pattern between ring B and C as well as the relative configurations of C-16 and C-17 (Figure 2). B

DOI: 10.1021/acs.orglett.9b02552 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Scheme 1. Proposed Biosynthetic Pathway of 1

of 1 in alignment media following our established protocol and fitted them with theoretic RDCs of DFT optimized geometries of 1a and 1b (Figure 4).8 As a result, 1a matched the experimental data well, showing a decent Q value (0.087), which was better than that of 1b (0.186). Thus, the planar structure and relative configuration of xylarichalasin A were determined to be 1a. To determine the absolute configuration, ECD calculation of 1 was performed on mPW1PW91/6-311G*/B3LYP/6-31G* (IEFPCM) level of theory. The calculated ECD curve of enantiomer 4R,5R,6S,8S,9R,13S,14R,16S,17S,19R,20S,21R matched the experimental ECD data well (Figure 5). Therefore, the stereochemistry of 1 can be fully assigned. Similar to the biosynthesis of curtachalasins C−E, xylarichalasin A might also be derived from the precursor 19,20-epoxycytochalasin C (Scheme 1). After the formation of rings D, E, and F as previously proposed steps,3b,c the CH3-11 and C-25 might be connected via a radical reaction, generating the seven membered ring B. Moreover, the two chlorine might be introduced at position C-10 and C-13 by halogenases.9 Cytochalasans are best known as cytotoxic compounds with actin filaments capping activity, which are considered as potential anticancer agents.1 Thus, we measured their cytotoxicity against five human cancer cell lines HL-60, A549, SMMC-7721, MCF-7, and SW480, following the established bioassay procedure for other obtained cytochalasans (Table 3).3b,10 Compound 1 showed significant cytotoxicity against SMMC-7721 and MCF-7 cell lines, which is better than the positive drug cisplatin. Against cell lines HL-60, A549, and SW480, however, it showed only moderate activities. In summary, xylarichalasin A represents a new class of cytochalasan with a unique benzo[7]annulene/pyrrolidine/ perhydroanthracene fused core structure. DFT 13C NMR calculation, RDC parameters, and ECD calculation were utilized to unambiguously determine its structure. As a result of bioassay, it showed the potential to be further investigated as an anticancer candidate.

Figure 4. Fitting theoretic (y) and experimental (x) RDCs for 1a and 1b.

Figure 5. ECD calculation (MeOH) of 1.

To assign the relative configuration of C-6 and confirm the deduced structure, we first calculated the 13C NMR of two possible structures 1a and 1b (Figure 3) on ωB97x-D/631G*//B3LYP/6-31G* level of theory with the reported procedure, using the scaling parameters for halogen-bearing carbons and ordinary carbons (Table 2).5 Because the linear formula in literature was optimized for the δC measured in CDCl3, we used the corresponding experimental data. The calculated 13C NMR of 1a matched experimental data very well, and the MAE and RMS values were lower than those of 1b. Moreover, the sDP4+ probabilities6 of 1a and 1b were 99.82% and 0.18%, respectively. This result showed that 1a is the correct structure for xylarichalasin A. Chemical shifts of 13C NMR reveal the chemical environment of carbons, while the anisotropic NMR parameters including RDCs code the global spatial information on structure.7 In our previous study, we successfully performed RDC measurement and calculation to assign the relative configurations of curtachalasins C and D.3c To double confirm the assigned structure, therefore, we measured the 1DCH RDCs C

DOI: 10.1021/acs.orglett.9b02552 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Table 3. Cytotoxicity of 1 against Five Human Cancer Cell Lines (IC50 ± SD in μM)

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compounds

HL-60

A-549

SMMC-7721

MCF-7

SW480

1 cisplatin

17.3 ± 1.6 2.0 ± 0.1

11.8 ± 0.2 13.2 ± 0.3

8.6 ± 0.2 12.7 ± 2.0

6.3 ± 0.1 23.3 ± 0.6

13.2 ± 0.3 18.0 ± 1.8

(4) Macías-Rubalcava, M. L.; Sánchez-Fernández, R. E. World J. Microbiol. Biotechnol. 2017, 33, 15. (5) Kutateladze, A. G.; Reddy, D. B. J. Org. Chem. 2017, 82, 3368− 3381. (6) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015, 80, 12526−12534. (7) (a) Li, G. W.; Liu, H.; Qiu, F.; Wang, X. J.; Lei, X. X. Nat. Prod. Bioprospect. 2018, 8, 279−295. (b) Troche-Pesqueira, E.; Anklin, C.; Gil, R. R.; Navarro-Vázquez, A. Angew. Chem., Int. Ed. 2017, 56, 3660−3664. (8) (a) Lei, X.; Qiu, F.; Sun, H.; Bai, L.; Wang, W.-X.; Xiang, W.; Xiao, H. Angew. Chem., Int. Ed. 2017, 56, 12857−12861. (b) Liu, L. Y.; Sun, H.; Griesinger, C.; Liu, J. K. Nat. Prod. Bioprospect. 2016, 6, 41−48. (c) Liu, H.; Chen, P.; Li, X.-L.; Sun, H.; Lei, X. Magn. Reson. Chem. 2019, 1−7. (9) (a) Ferrara, M.; Perrone, G.; Gambacorta, L.; Epifani, F.; Solfrizzo, M.; Gallo, A. Appl. Environ. Microbiol. 2016, 82, 5631− 5641. (b) Zeng, J.; Zhan, J. ChemBioChem 2010, 11, 2119−2123. (c) Zeng, J.; Lytle, A. K.; Gage, D.; Johnson, S. J.; Zhan, J. Bioorg. Med. Chem. Lett. 2013, 23, 1001−1003. (10) (a) Wang, W.-X.; Feng, T.; Li, Z.-H.; Li, J.; Ai, H.-L.; Liu, J.-K. Tetrahedron Lett. 2019, 60, 150952. (b) Wang, W.-X.; Li, Z.-H.; He, J.; Feng, T.; Li, J.; Liu, J.-K. Fitoterapia 2019, 137, 104278. (c) Wang, W.-X.; Li, Z.-H.; Ai, H.-L.; Li, J.; He, J.; Zheng, Y.-S.; Feng, T.; Liu, J.K. Fitoterapia 2019, 137, 104253.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b02552.

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Experimental procedures; 1D/2D NMR, MS, IR, ECD spectra, and RDC measurement for compound 1 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] ORCID

Wen-Xuan Wang: 0000-0002-7391-2954 Xinxiang Lei: 0000-0002-5635-5375 Hong-Lian Ai: 0000-0002-6832-0970 Jing Li: 0000-0001-8133-507X Tao Feng: 0000-0002-1977-9857 Ji-Kai Liu: 0000-0001-6279-7893 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 31801789, 81773590, 31870513, 81872762) and the National Key Research and Development Program of China (No. 2017YFC1704007). We are grateful for the convenience for the HRMS and NMR measurements provided by the Analytical & Measuring Center, School of Pharmaceutical Sciences, South-Central University for Nationalities for the spectral analyses.

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DOI: 10.1021/acs.orglett.9b02552 Org. Lett. XXXX, XXX, XXX−XXX